Substituted 2-aminobenzamide caspase inhibitors and the use thereof

ABSTRACT

The present invention is directed to novel 2-aminobenzamide thereof, represented by the general Formula I:                    
     where R 1 -R 3 , X, and A—D are defined herein. The present invention also relates to the discovery that compounds having Formula I are potent inhibitors of caspases and apoptotic cell death. Therefore, the inhibitors of this invention can retard or block cell death in a variety of clinical conditions in which the loss of cells, tissues or entire organs occurs.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the filing date of U.S.Provisional Application No. 60/158,386, filed Oct. 12, 1999, and U.S.Provisional Application No. 60/124,675, filed Mar. 16, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the field of medicinal chemistry. In particular,the invention relates to substituted 2-aminobenzamides and analogs thatare inhibitors of caspases. The invention also relates to the use ofthese 2-aminobenzamides and analogs for reducing or treating apoptoticcell death and/or reducing interleukin 1-β production.

2. Description of Background Art

Organisms eliminate unwanted cells by a process variously known asregulated cell death, programmed cell death or apoptosis. Such celldeath occurs as a normal aspect of animal development as well as intissue homeostasis and aging (Glucksmann, A., Biol. Rev. CambridgePhilos. Soc. 26:59-86 (1951); Glucksmann, A., Archives de Biologie76:419-437 (1965); Ellis et al., Dev. 112:591-603 (1991); Vaux et al.,Cell 76:777-779 (1994)). Apoptosis regulates cell number, facilitatesmorphogenesis, removes harmful or otherwise abnormal cells andeliminates cells that have already performed their function.Additionally, apoptosis occurs in response to various physiologicalstresses, such as hypoxia or ischemia (PCT published application WO96/20721).

There are a number of morphological changes shared by cells experiencingregulated cell death, including plasma and nuclear membrane blebbing,cell shrinkage (condensation of nucleoplasm and cytoplasm), organellerelocalization and compaction, chromatin condensation and production ofapoptotic bodies (membrane enclosed particles containing intracellularmaterial) (Orrenius, S., J. Internal Medicine 237:529-536 (1995)).

Apoptosis is achieved through an endogenous mechanism of cellularsuicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowenand Lockshin, eds., Chapman and Hall (1981), pp. 9-34). A cell activatesits internally encoded suicide program as a result of either internal orexternal signals. The suicide program is executed through the activationof a carefully regulated genetic program (Wylie et al., Int. Rev. Cyt.68:251 (1980); Ellis et al., Ann. Rev. Cell Bio. 7:663 (1991)).Apoptotic cells and bodies are usually recognized and cleared byneighboring cells or macrophages before lysis. Because of this clearancemechanism, inflammation is not induced despite the clearance of greatnumbers of cells (Orrenius, S., J. Internal Medicine 237:529-536(1995)).

Mammalian interleukin-1β (IL-1β) plays an important role in variouspathologic processes, including chronic and acute inflammation andautoimmune diseases (Oppenheim, J. H. et. al. Immunology Today, 7, 45-56(1986)). IL-1β is synthesized as a cell associated precursor polypeptide(pro-IL-1β) that is unable to bind IL-1 receptors and is biologicallyinactive (Mosley et al., J. Biol. Chem. 262:2941-2944 (1987)). Byinhibiting conversion of precursor IL-1β to mature IL-1β, the activityof interleukin-1 can be inhibited. Interleukin-1β converting enzyme(ICE) is a protease responsible for the activation of interleukin-1β(IL-1β) (Thornberry, N. A., et al., Nature 356:768 (1992); Yuan, J., etal., Cell 75:641 (1993)). ICE is a substrate-specific cysteine proteasethat cleaves the inactive prointerleukin-1 to produce the mature IL-1.The genes that encode for ICE and CPP32 are members of the mammalianICE/Ced-3 family of genes which presently includes at least twelvemembers: ICE, CPP32/Yama/Apopain, mICE2, ICE4, ICH1, TX/ICH-2, MCH2,MCH3, MCH4, FLICE/MACH/MCH5, ICE-LAP6 and ICE_(re1)III. The proteolyticactivity of this family of cysteine proteases, whose active site (acysteine residue) is essential for ICE-mediated apoptosis, appearscritical in mediating cell death (Miura et al., Cell 75:653-660 (1993)).This gene family has recently been named caspases (Alnernri, E. S. et.al. Cell, 87, 171 (1996), and Thomberry, N. A. et. al. J. Biol. Chem.272, 17907-17911 (1997)) and divided into three groups according to itsknown functions. Table 1 summarizes these known caspases.

TABLE 1 Enzyme* Group I: mediators of inflammation Caspase-1 (ICE)Caspase-4 (ICE_(rel)-II, TX, ICH-2) Caspase-5 (ICE_(rel)-III, TY) GroupII: effectors of apoptosis Caspase-2 (ICH-1, mNEDD2) Caspase-3 (apopain,CPP-32, YAMA) Caspase-7 (Mch-3, ICE-LAP3, CMH-1) Group III: activatorsof apoptosis Caspase-6 (Mch2) Caspase-8 (MACH, FLICE, Mch5) Caspase-9(ICE-LAP6, Mch6) Caspase-10

IL-1 is also a cytokine involved in mediating a wide range of biologicalresponses including inflammation, septic shock, wound healing,hematopoiesis and growth of certain leukemias (Dinarello, C. A., Blood77:1627-1652 (1991); diGiovine et al., Immunology Today 11:13 (1990)).

Many potent caspase inhibitors have been prepared based on the peptidesubstrate structures of caspases. However, in contrast to their potencyin vitro, no inhibitors with good efficacy (IC₅₀<1 μM) in whole-cellmodels of apoptosis have been reported (Thomberry, N. A. Chem. Biol.5:R97-103 (1998)). Therefore the need exists for cell death inhibitorsthat are efficacy in whole-cell models of apoptosis and active in animalmodel of apoptosis. These inhibitors thus can be employed as therapeuticagents to treat disease states in which regulated cell death and thecytokine activity of IL-1 play a role.

WO 93/05071 discloses peptide ICE inhibitors with the formula:

Z—Q ₂-Asp-Q ₁

wherein Z is an N-terminal protecting group; Q₂ is 0 to 4 amino acidssuch that the sequence Q₂-Asp corresponds to at least a portion of thesequence Ala-Tyr-Val-His-Asp; Q₁ comprises an electronegative leavinggroup.

WO 96/03982 discloses aspartic acid analogs as ICE inhibitors with theformula:

wherein R₂ is H or alkyl; R₃ is a leaving group such as halogen; R₁ isheteroaryl-CO or an amino acid residue.

U.S. Pat. No. 5,585,357 discloses peptidic ketones as ICE inhibitorswith the formula:

wherein n is 0-2; each AA is independently L-valine or L-alanine; R₁ isselected from the group consisting of N-benzyloxycarbonyl and othergroups; R₈, R₉, R₁₀ are each independently hydrogen, lower alkyl andother groups.

Mjalli et al. (Bioorg. Med. Chem. Lett., 3, 2689-2692, 1993) report thepreparation of peptide phenylalkyl ketones as reversible inhibitors ofICE, such as:

Thornberry et al. (Biochemistry, 33, 3934-3940, 1994) report theirreversible inactivation of ICE by peptide acyloxymethyl ketones:

wherein Ar is COPh-2,6-(CF₃)₂, COPh-2,6-(CH₃)₂, Ph-F₅ and other groups.

Dolle et al. (J. Med. Chem. 37, 563-564, 1994) report the preparation ofP₁ aspartate-based peptide α-((2,6-dichlorobenzoyl)oxy)methyl ketones aspotent time-dependent inhibitors of ICE, such as:

Mjalli et al. (Bioorg. Med Chem. Lett., 4, 1965-1968, 1994) report thepreparation of activated ketones as potent reversible inhibitors of ICE:

wherein X is NH(CH₂)₂, OCO(CH₂)₂, S(CH₂)₃ and other groups.

Dolle et al. (J. Med. Chem. 37, 3863-3866, 1994) report the preparationof α-((1-phenyl-3-(trifluoromethyl)-pyrazol-5-yl)oxy)methyl ketones asirreversible inhibitor of ICE, such as:

Mjalli et al. (Bioorg Med. Chem. Lett., 5, 1405-1408, 1995) reportinhibition of ICE by N-acyl-Aspartic acid ketones:

wherein XR₂ is NH(CH₂)₂Ph, OCO(CH₂)₂cyclohexyl and other groups.

Mjalli et al. (Bioorg Med Chem. Lett., 5, 1409-1414, 1995) reportinhibition of ICE by N-acyl-aspartyl aryloxymethyl ketones, such as:

Dolle et al. (J. Med. Chem. 38, 220-222, 1995) report the preparation ofaspartyl α-((diphenylphosphinyl)oxy)methyl ketones as irreversibleinhibitors of ICE, such as:

Graybill et al. (Bioorg. Med. Chem. Lett., 7, 41-46, 1997) report thepreparation of α-((tetronoyl)oxy)- and α-((tetramoyl)oxy)methyl ketonesas inhibitors of ICE, such as:

Semple et al. (Bioorg. Med. Chem. Lett., 8, 959-964, 1998) report thepreparation of peptidomimetic aminomethylene ketones as inhibitors ofICE, such as:

SUMMARY OF THE INVENTION

The invention relates to compound of Formulae I, II and III:

wherein

R₁ is an optionally substituted alkyl or hydrogen;

R₃ is an N-protecting group;

R₂ is hydrogen or optionally substituted alkyl;

A is CR₆ or nitrogen;

B is CR₇ or nitrogen;

C is CR₈ or nitrogen;

D is CR₉ or nitrogen;

provided that not more than two of A, B, C or D is nitrogen; and R₆-R₉independently are hydrogen, halo, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀aryl(C₁-C₆) C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, C₁-C₆hydroxyalkyl, nitro, amino, cyano, C₁-C₆ acylamino, hydroxy, C₁-C₆acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy; or

one of R₆ and R₇, or R₁ and R₈, or R₈ and R₉ are taken together with thecarbon atoms to which they are attached to form a carbocycle orheterocycle;

E is CR₁₄, nitrogen, oxygen or sulfur;

F is CR₁₅, nitrogen, oxygen or sulfur;

G is C₁₆, nitrogen, oxygen or sulfur;

provided that only one of E, F, G is nitrogen, oxygen or sulfur; whereR₁₄-R₁₆ are independently hydrogen, halo, C₁-C₆ haloalkyl, C₆-C₁₀ aryl,C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl,C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆ acylamino, hydroxy, C₁-C₆acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy; or

one of R₁₄ and R₁₅, or R₁₅ and R₁₆, are taken together with the carbonatoms to which they are attached to form a carbocycle or heterocycle;

Q represents an optionally substituted saturated or partially saturatedcarbocycle or heterocycle;

X is a peptide of 1-4 amino acids or a bond; and

Y is a peptide of 1-4 amino acids or a bond.

The invention relates to the discovery that the compounds represented byFormulae I, II and III are inhibitors of caspases. The invention alsorelates to the use of the compounds of the invention for reducing,preventing or treating maladies in which apoptotic cell death is eithera causative factor or a result. Examples of uses for the presentinvention include protecting the nervous system following focal ischemiaand global ischemia; treating neurodegenerative disorders such asAlzheimer's disease, Huntington's Disease, prion diseases, Parkinson'sDisease, multiple sclerosis, amyotrophic lateral sclerosis, ataxia,telangiectasia, and spinobulbar atrophy; treating heart diseaseincluding myocardial infarction, congestive heart failure andcardiomyopathy; treating retinal disorders; treating autoimmunedisorders including lupus erythematosus, rheumatoid arthritis, type Idiabetes, Sjögren's syndrome and glomerulonephritis; treating polycystickidney disease and anemia/erythropoiesis; treating immune systemdisorders, including AIDS and SCIDS; treating or ameliorating sepsis ormulti-organ failure in an animal; reducing or preventing cell, tissueand organ damage during transplantation; reducing or preventing cellline death in industrial biotechnology; reducing or preventing alopecia(hair loss); reducing the premature death of skin cells; treating orameliorating apoptotic cell death in acute pancreatitus; treating orpreventing the inflammatory response in psoriasis or inflammatory boweldisease; and treating or ameliorating organ apoptosis after burn injury.

The present invention provides pharmaceutical compositions comprising acompound of Formulae I, II and III in an effective amount to reduceapoptotic cell death in an animal.

The present invention also provides preservation or storage solutionsfor mammalian organs or tissue, or growth media for mammalian or yeastcells, wherein an effective amount of a compound of Formula I, II andIII is included in said solutions or media in order to reduce apoptoticcell death in said organs, tissue or cells.

The invention also relates to the use of caspase inhibitors fortreating, ameliorating, and preventing non-cancer cell death duringchemotherapy and radiation therapy and for treating and ameliorating theside effects of chemotherapy and radiation therapy of cancer.

In particular, the invention relates to a method of treating,ameliorating or preventing oral mucositis, gastrointestinal mucositis,bladder mucositis, proctitis, bone marrow cell death, skin cell deathand hair loss resulting from chemotherapy or radiation therapy of cancerin an animal, comprising administering to the animal in need thereof aneffective amount of a caspase inhibitor.

Detailed Description of the Invention

The inhibitors of caspases and apoptotic cell death of the presentinvention are compounds having the general Formulae I, II and III:

or pharmaceutically acceptable salts or prodrugs thereof, wherein:

R₁ is an optionally substituted alkyl or hydrogen;

R₃ is a N-protecting group including t-butyloxycarbonyl, acetyl, andbenzyloxycarbonyl;

R₂ is hydrogen or optionally substituted alkyl;

A is CR₆ or nitrogen;

B is CR₇ or nitrogen;

C is CR₈ or nitrogen;

D is CR₉ or nitrogen; provided that not more than two of A, B, C or D isnitrogen; and R₆-R₉ independently are hydrogen, halo, C₁-C₆ haloalkyl,C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy;or

one of R₆ and R₇, or R₇ and R₈, or R₈ and R₉ are taken together with thecarbon atoms to which they are attached to form a carbocycle orheterocycle;

E is C₁₄, nitrogen, oxygen or sulfur;

F is C₁₅, nitrogen, oxygen or sulfur;

G is C₁₆, nitrogen, oxygen or sulfur; provided that only one of E, F, Gis nitrogen, oxygen or sulfur; where R₁₄-R₁₆ are independently hydrogen,halo, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl,nitro, amino, cyano, C₁-C₆ acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆alkoxy, alkylthio, or carboxy; or

one of R₁₄ and R₁₅, or R₁₅ and R₁₆ are taken together with the carbonatoms to which they are attached to form a carbocycle or heterocycle;

Q represents an optionally substituted saturated or partially saturatedcarbocycle or heterocycle;

X is a peptide of 1-4 amino acids or a bond; And

Y is a peptide of 1-4 amino acids or a bond. Where X or Y is one aminoacid, it may be any one of the common 20 amino acids e.g., Ala, Val,Leu, Ile, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asp, Asn, Glu,Asn, Lys, Arg and His. Where X is a peptide, it may be Asp-Glu, Asp-Ala,Asp-Phe, Val-Glu, Leu-Glu, Thr-Glu, Ile-Glu, Tyr-Glu, Trp-Glu. Where Yis a peptide, it may be Glu-His, Glu-Ile, Glu-Thr, Glu-Val, Glu-Phe,Thr-His, Val-His, Ala-His and Glu-Pro.

With respect to R₁, preferred alkyl groups are C₁₋₆ alkyl groups, e.g.methyl, ethyl, propyl, isopropyl, isobutyl, pentyl and hexyl groups; andsubstituted C₁₋₆alkyl groups, e.g. CH₂OCH₃ and CH₂OCOCH₃ (AM).

The invention relates to the discovery that the compounds represented byFormulae I, II and III are inhibitors of caspases. These inhibitors slowor block cell death in a variety of clinical conditions and industrialapplications in which the loss of cells, tissues or entire organsoccurs. Therefore, the invention is also related to methods of treating,preventing or reducing conditions in which apoptosis plays a role. Theseconditions are more fully described below.

The methods comprise administering to an animal in need of suchtreatment an inhibitor of the present invention, or a pharmaceuticallyacceptable salt or prodrug thereof, in an amount effective to inhibitapoptotic cell death.

Preferred embodiments of the compounds of Formulae I, II and III thatmay be employed as inhibitors of caspases are represented by Formula IV:

or pharmaceutically acceptable salts or prodrugs thereof wherein R₁ R₂-,R₆-R₉ and X are as defined previously with respect to Formula I.

Examples of bridges formed by R₆ and R₇, or R₇ and R₈, or R₈ and R₉taken together are —OCH₂O—, —OCF₂O—, —(CH₂)₃—, —(CH₂)₄—, —OCH₂CH₂O—,—CH₂N(R₁₃)CH₂—, —CH₂CH₂N(R₁₃)CH₂—, —CH₂N(R₁₃)CH₂CH₂— and —CH═CH—CH═CH—;where R₁₃ is hydrogen, alkyl or cycloalkyl.

R₁₀ is hydrogen, C₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₆-C₁₀aryl, C₄-C₇ cycloalkyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, benzyloxy, substitutedbenzyloxy, or NR₁₁R₁₂, wherein R₁₁ and R₁₂ independently are hydrogen,C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₆-C₁₀aryl(C₁-C₆)alkyl, or R₁₁ and R₁₂ are combined to form a heterocyclicring system including pyrrolidine, piperidine, piperazine, ormorpholine.

Preferred R₁ is H, Me, Et, t-Bu or AM. Preferred R₂ is fluoromethyl,acyloxymethyl, arylacyloxymethyl and aminomethyl. Preferred R₁₀ isbenzyloxy and substituted benzyloxy. Preferred X is a peptide of 1-2amino acids or a bond.

Exemplary preferred inhibitors of caspases having Formulae I-IV include,without limitation:

2-(Z-amino)benzoyl-Asp-fmk,

2-(Z-amino)-3-methylbenzoyl-Asp-fmk,

2-(Z-amino)-3,5-dimethylbenzoyl-Asp-fmk,

2-(Z-amino)-4-chlorobenzoyl-Asp-fmk,

2-(Z-amino)-5-chlorobenzoyl-Asp-fmk,

2-(Z-amino)-5-fluorobenzoyl-Asp-fmk,

2-(Z-amino)-6-fluorobenzoyl-Asp-fmk,

cis-2-(Z-amino)-cyclohexanecarboxyl-Asp-fmk,

2-(Z-amino)-5-methylbenzoyl-Asp-fmk,

2-(Z-amino)-6-methylbenzoyl-Asp-fmk,

2-(Z-amino)-6-chlorobenzoyl-Asp-fmk,

2-(Z-amino)-3-methoxybenzoyl-Asp-fmk,

3-(Z-amino)thiophene-2-carboxyl-Asp-fmk,

3-(methoxycarbonylamino)thiophene-2-carboxyl-Asp-fmk,

cis-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,

trans-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,

2-(Z-amino)benzoyl-Asp-DCB-methylketone,

methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk,

Z-Glu-(2-aminobenzoyl)-Asp-fmk, and

Z-Val-(2-aminobenzoyl)-Asp-fmk.

where Z is benzyloxycarbonyl, fmk is fluoromethylketone and DCB is2,6-dichlorobenzoyloxy.

Useful aryl groups are C₆₋₁₄ aryl, especially C₆₋₁₀ aryl. Typical C₆₋₁₄aryl groups include phenyl, naphthyl, phenanthrenyl, anthracenyl,indenyl, azulenyl, biphenyl, biphenylenyl and fluorenyl groups.

Useful cycloalkyl groups are C₃₋₈ cycloalkyl. Typical cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl.

Useful saturated or partially saturated carbocyclic groups arecycloalkyl groups as defined above, as well as cycloalkenyl groups, suchas cyclopentenyl, cycloheptenyl and cyclooctenyl.

Useful halo or halogen groups include fluorine, chlorine, bromine andiodine.

Useful alkyl groups include straight-chained and branched C₁₋₁₀ alkylgroups, more preferably C₁₋₆ alkyl groups. Typical C₁₋₁₀ alkyl groupsinclude methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl,3-pentyl, hexyl and octyl groups. Also contemplated is a trimethylenegroup substituted on two adjoining positions on the benzene ring of thecompounds of the invention.

Useful arylalkyl groups include any of the above-mentioned C₁₋₁₀, alkylgroups substituted by any of the above-mentioned C₆₋₁₄ aryl groups.Useful values include benzyl, phenethyl and naphthylmethyl.

Useful haloalkyl groups include C₁₋₁₀ alkyl groups substituted by one ormore fluorine, chlorine, bromine or iodine atoms, e.g. fluoromethyl,difluoromethyl, trifluoromethyl, pentafluoroethyl, 1,1-difluoroethyl,chloromethyl, chlorofluoromethyl and trichloromethyl groups.

Useful alkoxy groups include oxygen substituted by one of the C₁₋₁₀alkyl groups mentioned above.

Useful alkylthio groups include sulphur substituted by one of the C₁₋₁₀alkyl groups mentioned above. Also included are the sulfoxides andsulfones of such alkylthio groups.

Useful acylamino groups are any C₁₋₆ acyl (alkanoyl) attached to anamino nitrogen, e.g. acetamido, propionamido, butanoylamido,pentanoylamido, hexanoylamido as well as aryl-substituted C₂₋₆substituted acyl groups.

Useful acyloxy groups are any C₁₋₆ acyl (alkanoyl) attached to an oxy(—O—) group, e.g. formyloxy, acetoxy, propionoyloxy, butanoyloxy,pentanoyloxy, hexanoyloxy and the like.

Useful arylacyloxy groups include any of the aryl groups mentioned abovesubstituted on any of the acyloxy groups mentioned above, e.g.2,6-dichlorobenzoyloxy, 2,6-difluorobenzoyloxy and2,6-di-(trifluoromethyl)-benzoyloxy groups.

Useful amino groups include —NH₂, —NHR₁₁, and —NR₁₁R₁₂, wherein R₁₁ andR₁₂ are C₁₋₁₀ alkyl or cycloalkyl groups as defined above.

Useful saturated or partially saturated heterocyclic groups includetetrahydrofuranyl, pyranyl, piperidinyl, piperizinyl, pyrrolidinyl,imidazolidinyl, imidazolinyl, indolinyl, isoindolinyl, quinuclidinyl,morpholinyl, isochromanyl, chromanyl, pyrazolidinyl pyrazolinyl,tetronoyl and tetramoyl groups.

Useful heteroaryl groups include any one of the following: thienyl,benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxanthiinyl, 2H-pyrrolyl,pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl,purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl,naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, carbazolyl,β-carbolinyl, phenanthridinyl, acrindinyl, perimidinyl, phenanthrolinyl,phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl,phenoxazinyl, 1,4-dihydroquinoxaline-2,3-dione, 7-aminoisocoumarin,pyrido[1,2-a]pyrimidin-4-one, 1,2-benzoisoxazol-3-yl, benzimidazolyl,2-oxindolyl and 2-oxobenzimidazolyl. Where the heteroaryl group containsa nitrogen atom in a ring, such nitrogen atom may be in the form of anN-oxide, e.g. a pyridyl N-oxide, pyrazinyl N-oxide, pyrimidinyl N-oxideand the like.

Optional substituents include one or more alkyl; halo; haloalkyl;cycloalkyl; aryl optionally substituted with one or more lower alkyl,halo, haloalkyl or heteroaryl groups; aryloxy optionally substitutedwith one or more lower alkyl, halo, haloalkyl or heteroaryl groups;aralkyl; heteroaryl optionally substitued with one or more lower alkyl,haloalkyl and aryl groups; heteroaryloxy optionally substitued with oneor more lower alkyl, haloalkyl and aryl groups; alkoxy; alkylthio;arylthio; amino; acyloxy; arylacyloxy optionally substitued with one ormore lower alkyl, halo alkyl and aryl groups; Diphenylphosphinyloxyoptionally substituted with one or more lower alkyl, halo or haloalkylgroups; heterocyclo optionally substitued with one or more lower alkyl,halo alkyl and aryl groups; heterocycloalkyloxy optionally substituedwith one or more lower alkyl, halo alkyl and aryl groups; partiallyunsaturated heterocycloalkyl optionally substitued with one or morelower alkyl, halo alkyl and aryl groups; or partially unsaturatedheterocycloalkyloxy optionally substitued with one or more lower alkyl,halo alkyl and aryl groups. Particular examples of such optionalsubstituents that may be present at R₂ include, without limitation,3-pyrazolyloxy optionally substituted at the 2, 4 and 5-positions withlower alkyl; 3-(1-phenyl-3-trifluoromethyl)pyrazolyloxy;2,6-di(trifluoromethyl)benzoyloxy; 2,6-dimethylbenzoyloxy,pentafluorophenoxy; 2,6-dichlorobenzoyloxy;2-(3-(2-imidazolyl)naphthyl)oxy; diphenylphosphinyloxy; tetronyloxy; andtetramoyloxy.

Certain of the compounds of the present invention may exist asstereoisomers including optical isomers. The invention includes allstereoisomers and both the racemic mixtures of such stereoisomers aswell as the individual enantiomers that may be separated according tomethods that are well known to those of ordinary skill in the art.

Examples of pharmaceutically acceptable addition salts include inorganicand organic acid addition salts such as hydrochloride, hydrobromide,phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate,mandelate and oxalate; and inorganic and organic base addition saltswith bases such as sodium hydroxy and Tris(hydroxymethyl)aminomethane(TRIS, tromethane).

Examples of prodrugs include compounds of Formulae I-IV wherein R₁ is analkyl group or substituted alkyl group such as CH₂OCH₃ and CH₂OCOCH₃ (AMester).

The invention is also directed to a method for treating disordersresponsive to the inhibition of caspases in animals suffering thereof.Particular preferred embodiments of compounds for use in the method ofthis invention are represented by previously defined Formulae I-IV.

The compounds of this invention may be prepared using methods known tothose skilled in the art. Specifically, compounds with Formulae I-IV canbe prepared as illustrated by exemplary reactions in Scheme 1. Theintermediate 1 was prepared according to Revesz et al. (TetrahedronLett. 35, 9693-9696, 1994). Coupling of 1 with a N-protected2-aminobenzoic acid, which is either commercially available or which canbe prepared from a commercially available 2-aminobenzoic acid, such as2-Z-aminobenzoic acid, gave amide 2. Oxidation of 2 by Dess-Martinreagent according to Revesz et al. (Tetrahedron Lett. 35, 9693-9696,1994) gave 3. Acid catalyzed cleavage of the ester gave the free acid 4.

An important aspect of the present invention is the discovery thatcompounds having Formnulae I-IV are inhibitors of caspases. Therefore,these inhibitors are expected to slow or block cell death in a varietyof clinical conditions in which the loss of cells, tissues or entireorgans occurs.

The cell death inhibitors of the present invention can be used to reduceor prevent cell death in the nervous system (brain, spinal cord, andperipheral nervous system) under various conditions of ischemia andexcitotoxicity, including, but not limited to, focal ischemia due tostroke and global ischemia due to cardiac arrest, as well as spinal cordinjury (Emery et al. J. Neurosurgery, 89:911-920 (1998)). One particularusage is to treat the effects of oxygen deprivation which can occurduring the birth of infants in high-risk labors or drowning. The celldeath inhibitors can also be used to reduce or prevent cell death in thenervous system due to traumatic injury (such as head trauma), viralinfection or radiation-induced nerve cell death (for example, as aside-effect of cancer radiotherapy). The cell death inhibitors can alsobe used to reduce or prevent cell death in a range of neurodegenerativedisorders, including but not limited to Alzheimer's disease (Mattson etal. Brain Res. 807:167-176 (1998)), Huntington's Disease, Parkinson'sDisease, multiple sclerosis, amyotrophic lateral sclerosis, andspinobulbar atrophy. The in vivo neuroprotective properties of celldeath inhibitors of the invention can be tested in a rat transient focalbrain ischemia model (Xue et al., Stroke 21:166 (1990)). The cell deathinhibitors may also be used to treat or ameliorate cell death in acutebacterial meningitis.

The cell death inhibitors of the invention can be used to reduce orprevent cell death in any condition which potentially results in thedeath of cardiac muscle (Black et al., J. Mol. Cel. Card. 30:733-742(1998) and Maulik et al. Free Radic. Biol. Med. 24:869-875 (1998)). Thisincludes myocardial infarction due to myocardial ischemia andreperfusion, congestive heart failure and cardiomyopathy. One particularapplication is to reduce or prevent myocardial cell death as occurs incertain viral infections of the heart.

The in vivo activity of the cell death inhibitors of the invention canbe tested using the “mouse liver apoptosis” model described by Rodriguezet al. (Rodriguez et al., J. Exp. Med., 184:2067-2072 (1996)). In thismodel, mice are treated intravenously,(IV) with an antiFas antibodywhich induces massive apoptosis in the liver and other organs, leadingto generalized organ failure and death. This model is useful forindirectly testing the systemic bioavailability of the cell deathinhibitors of the invention, as well as their in vivo anti-apoptoticproperties. The cell death inhibitors of the invention therefore can beused to reduce or prevent apoptosis of liver cells (Jones et al.Hepatology 27:1632-42 (1998)) such as in sepsis (Jaeschke et al. J.Immunol. 160:3480-3486 (1998)) and hereditary tyrosinemia type 1 (HT1)(Kubo et al. Prov. Natl. Acad. Sci. USA, 95:9552-9557 (1998). The celldeath inhibitors of the invention also can be used to treat hepatitis(Suzuki, Proc. Soc. Exp. Biol. Med. 217:450-454 (1998)); treat orameliorate apoptotic cell death in acute pancreatitus; and treat orameliorate organ apoptosis after burn injury.

The cell death inhibitors of the invention can be used to reduce orprevent cell death of retinal neurons (Kermer et al. J. Neurosci.18:4656-4662 (1998) and Miller et al. Am. J. Vet. Res. 59: 149-152(1998)) as can occur in disorders which increase intraocular pressure(such as glaucoma) or retinal disorders associated with the agingprocess (such as age-related macular degeneration). The inhibitors canalso be used to treat hereditary degenerative disorders of the retina,such as retinitis pigmentosa.

The cell death inhibitors of the invention can also be used to reduce orprevent cell death in the kidney. This includes renal amyloidosis(Hiraoka et al. Nippon Jinzo Gakkai Shi, 40:276-83 (1998)), acute renalfailure (Lieberthal et al. Semin Nephrol. 18:505-518 (1998)), murinetubular epithelial cell death induced by cyclosporine A (Ortiz et al.Kidney International Supp. 68: S25-S29 (1998)) and HIV-inducednephropathy (Conaldi et al. J. Clin. Invest. 102:2041-2049 (1998)).

The cell death inhibitors of the invention can also be used to reduce orprevent cell death of buccal mucosa due to chronic alcohol ingestion(Slomiany et al. Biochem. Mol. Biol. Int. 45:1199-1209 (1998)).

The cell death inhibitors of the invention can also be used to reduce orprevent cell death in plants (Richberg et al. Curr. Opin. Plant Biol.1:480-485 (1998)), such as plant cell death due to pathogens (Pozo etal. Curr. Biol. 8:1129-1132 (1998) and Greenberg et al. Cell, 77:551-563(1994)).

The cell death inhibitors of the invention can also be used to reduce orprevent cell death due to radiation and ultraviolet-irradiation (Sheikhet al. Oncogene, 17:2555-2563 (1998)).

The cell death inhibitors of the invention can also be used to reduce orprevent apoptotic death of bone marrow cells in myelodysplasticsyndromes (MDS) (Mundle et al., Am. J. Hematol. 60:36-47 (1999)).

The cell death inhibitors of the invention can also be used to reduce orprevent premature death of cells of the immune system, and areparticularly useful in treating immune deficiency disorders, such asacquired immune deficiency syndrome (AIDS), severe combined immunedeficiency syndrome (SCIDS) and related diseases. The cell deathinhibitors can also be used to treat radiation-induced immunesuppression.

Transplantation of human organs and tissues is a common treatment fororgan failure. However, during the transplantation process, the donororgan or tissue is at risk for cell death since it is deprived of itsnormal blood supply prior to being implanted in the host. This ischemicstate can be treated with cell death inhibitors by infusion into thedonor organ or tissue, or by direct addition of the cell deathinhibitors to the organ/tissue storage medium. Cell death inhibitors mayalso be used to reduce or prevent cell death in the donor organ/tissueafter it has been transplanted to protect it from the effects ofreperfusion injury and/or effects of host immune cells which kill theirtargets by triggering apoptosis. The cytoprotective effects of celldeath inhibitors can also be used to prevent the death of human oranimal sperm and eggs used in in vitro fertilization procedures. Theseinhibitors can be used during the harvesting process and can also beincluded in the storage medium.

Mammalian cell lines, insect cells and yeast cells are commonly used toproduce large amounts of recombinant proteins (such as antibodies,enzymes or hormones) for industrial or medicinal use. The lifespan ofsome of these cell lines is limited due to growth conditions, the natureof the recombinant molecule being expressed (some are toxic) and otherunknown factors. The lifespans of industrial cell lines can be extendedby including these cell death inhibitors in the growth medium in aconcentration range of 1-100 μM.

The factors governing hair growth and loss are largely unknown. There issome evidence, however, that hair follicle regression (referred to ascatagen) may be due at least partially to apoptosis. Therefore, it iscontemplated that the cell death inhibitors of the present invention canbe used to treat hair loss that occurs due to various conditions,including but not limited to male-pattern baldness, radiation-induced orchemotherapy-induced hair loss, and hair loss due to emotional stress.There is also evidence that apoptosis may play a role in the loss ofhair color. Therefore, it is contemplated that the cell death inhibitorsof the present invention can also be used in treating or preventingcases of premature graying of the hair.

The death of skin epithelial cells can occur after exposure to highlevels of radiation, heat or chemicals. It is contemplated that the celldeath inhibitors of the present invention can be used to treat, reduceor prevent this type of skin damage. In one particular application, thecell death inhibitors can be applied as part of a topical formulation,e.g. an ointment, to treat acute over-exposure to the sun and to preventblistering and peeling of the skin.

Goldberg et al. (Nature Genetics 13:442-449 (1996)) reported recentlythat huntingtin, a protein product of Huntington's disease (HD) gene,can be cleaved by CPP32 but not ICE. The mutation underlying HD is anexpansion of a CAG trinucleotide at the 5′ end of the HD gene. Thetrinucleotide expansion exceeding 36 repeats is associated with theclinical presentation of HD. The CAG expansion promotes cleavage ofhuntingtin by CPP32, thus links the role of CPP32 in the apoptotic celldeath in HD. Compounds of the present invention with CPP32 inhibitingactivity will be useful in blocking CPP32 induced apoptotic cell death,thus in preventing and treating HD and other disorders characterized byexpansion of trinucleotide repeats such as myotonic dystrophy, fragile Xmental retardation, spinobulbar muscular atrophy, spinocerebellar atoxiatype I and Dentato-Rubro pallidoluysian atrophy.

The invention relates to a method of treating, ameliorating orpreventing oral mucositis, gastrointestinal mucositis, bladdermucositis, proctitis, bone marrow cell death, skin cell death and hairloss resulting from chemotherapy or radiation therapy of cancer in ananimal, comprising administering to the animal in need thereof aneffective amount of a caspase inhibitor.

When animals are treated with chemotherapeutic agents and/or radiationto kill cancer cells, an unwanted side effect is the apoptotic death ofrapidly dividing non-cancer cells. Such non-cancer cells include cellsof the gastrointestinal tract, skin, hair, and bone marrow cells.According to the present invention, caspase inhibitors are administeredto such non-cancer cells to prevent apoptosis of such cells. In apreferred embodiment, the caspase inhibitors are administered locally,e.g. to the gastrointestinal tract, mouth, skin or scalp to preventapoptosis of the gastrointestinal, mouth, skin or hair cells butallowing for the death of the cancer cells. Thus, in one example, it ispossible to treat brain cancer with chemotherapy or radiation therapyand protect the outer skin, hair cells, gastrointestinal tract and bonemarrow by local administration of a caspase inhibitor. In the case oforal mucositis, the caspase inhibitor can be applied, for example, inthe form of a mouth wash or mouth rinse, in a gel, or in the form of anoral slow release lozenge to prevent activation of caspases andapoptotic cell death induced by the chemotherapeutic agent or byradiation. In the case of gastrointestinal mucositis, the caspaseinhibitor can be applied in a form such that it is not absorbedsystemically or in a form that coats the surface of the gastrointestinaltract, or a suppository formulation for the treatment ofgastrointestinal mucositis. In the case of proctitis, the capsaseinhibitor may be applied as part of an enema or suppository. In the caseof bladder mucositis, the caspase inhibitor may be applied though abladder catheter. For prevention of radiation or chemotherapy-inducedhair loss, the caspase inhibitor can be applied, for example, to thescalp in the form of a hair rinse, hair gel, shampoo or hairconditioner. Importantly, the caspase inhibitor can be applied prior tothe administration of the chemotherapeutic agent or radiation, thuspreventing the onset of the damaging effects of the chemotherapeuticagent or radiation to the normal cells.

The cell death inhibitors of the present invention may also be used totreat or prevent the inflammatory response in psoriasis or inflammatorybowel disease.

Compositions within the scope of this invention include all compositionswherein the compounds of the present invention are contained in anamount which is effective to achieve its intended purpose. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of each component is within the skill of the art. Typically, thecompounds may be administered to mammals, e.g. humans, orally at a doseof 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceuticallyacceptable salt thereof, per day of the body weight of the mammal beingtreated for apoptosis-mediated disorders, e.g., neuronal cell death,heart disease, retinal disorders, polycystic kidney disease, immunesystem disorders and sepsis. Preferably, about 0.01 to about 10 mg/kg isorally administered to treat or prevent such disorders. Forintramuscular injection, the dose is generally about one-half of theoral dose. For example, for treatment or prevention of neuronal celldeath, a suitable intramuscular dose would be about 0.0025 to about 25mg/kg, and most preferably, from about 0.01 to about 5 mg/kg.

The unit oral dose may comprise from about 0.01 to about 50 mg,preferably about 0.1 to about 10 mg of the compound. The unit dose maybe administered one or more times daily as one or more tablets eachcontaining from about 0.1 to about 10, conveniently about 0.25 to 50 mgof the compound or its solvates.

In a topical formulation, the compound may be present at a concentrationof about 0.01 to 100 mg per gram of carrier. In a preferred embodiment,the compound is present at a concentration of about 0.07-1.0 mg/ml, morepreferably, about 0.1-0.5 mg/ml, most preferably, about 0.4 mg/ml.

In addition to administering the compound as a raw chemical, thecompounds of the invention may be administered as part of apharmaceutical preparation containing suitable pharmaceuticallyacceptable carriers comprising excipients and auxiliaries whichfacilitate processing of the compounds into preparations which can beused pharmaceutically. Preferably, the preparations, particularly thosepreparations which can be administered orally or topically and which canbe used for the preferred type of administration, such as tablets,dragees, slow release lozenges and capsules, mouth rinses and mouthwashes, gels, liquid suspensions, hair rinses, hair gels, shampoos andalso preparations which can be administered rectally, such assuppositories, as well as suitable solutions for administration byinjection, topically or orally, contain from about 0.01 to 99 percent,preferably from about 0.25 to 75 percent of active compound(s), togetherwith the excipient.

Also included within the scope of the present invention are thenon-toxic pharmaceutically acceptable salts of the compounds of thepresent invention. Acid addition salts are formed by mixing a solutionof the particular cell death inhibitors of the present invention with asolution of a pharmaceutically acceptable non-toxic acid such ashydrochloric acid, fumaric acid, maleic acid, succinic acid, aceticacid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalicacid, and the like. Basic salts are formed by mixing a solution of theparticular cell death inhibitors of the present invention with asolution of a pharmaceutically acceptable non-toxic base such as sodiumhydroxide, potassium hydroxide, choline hydroxide, sodium carbonate Trisand the like.

The pharmaceutical compositions of the invention may be administered toany animal which may experience the beneficial effects of the compoundsof the invention. Foremost among such animals are mammals, e.g., humans,although the invention is not intended to be so limited.

The caspase inhibitors and pharmaceutical compositions thereof may beadministered by any means that achieve their intended purpose. Forexample, administration may be by parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, buccal, intrathecal,intracranial, intranasal or topical routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired. In general, the caspase inhibitorsare administered locally to the tissues that are to be protected fromapoptosis and separately from the chemotherapeutic agent. For example,cisplatin may be administered by i.v. injection to treat a cancer suchas brain, lung, breast, liver, kidney, pancreatic, ovarian, prostaticcancer, and the caspase inhibitor administered locally to treat,ameliorate, or prevent apototic cell death in the mouth orgastrointestinal tract, such as a mouth wash for the treatment of oralmucositis; and IV injectable aqueous solution for the treatment of bonemarrow cell death; and an oral formulation suitable for coating thegastrointestinal surfaces or an emema or suppository formulation for thetreatment of gastrointestinal mucositis including proctitis. The caspaseinhibitors may also be applied through a bladder catheter for thetreatment, amelioration or prevention of bladder mucositis.Alternatively or concurrently, the caspase inhibitors may be appliedtopically to the skin and/or scalp to treat, ameliorate or preventapoptotic cell death of hair and skin cells. In a further embodiment,the chemotherapeutic agent or radiation may be applied locally to treata localized cancer such as brain, lung, breast, liver, kidney,pancreatic, ovarian, prostatic cancer, and the caspase inhibitoradministered systemically, e.g. by i.v. injection, to treat, ameliorateor prevent apoptotic cell death of the gastrointestinal tract cells,mouth epithelial cells, bone marrow cells, skin cells and hair cells. Inthe case of oral mucositis in brain cancer treatment, for example, acaspase inhibitor that does not penetrate the blood-brain barrier can beapplied, for example, systemically by i.v. injection followed byirradiation of the brain tumor. This would protect the oral mucosa fromthe harmful effects of radiation but the caspase inhibitor would notprotect the brain tumor from the therapeutic effects of radiation.Importantly, the caspase inhibitor can be applied prior toadministration of the radiation, thus preventing the onset of thedamaging effects of the radiation to the normal mucosa cells.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, forexample lactose or sucrose, mannitol or sorbitol, cellulose preparationsand/or calcium phosphates, for example tricalcium phosphate or calciumhydrogen phosphate, as well as binders such as starch paste, using, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose,sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare, above all, flow-regulating agents and lubricants, for example,silica, talc, stearic acid or salts thereof, such as magnesium stearateor calcium stearate, and/or polyethylene glycol. Dragee cores areprovided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated saccharide solutions maybe used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropymethyl-cellulose phthalate, are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize combinations of activecompound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin. In addition, stabilizers may be added.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories, which consist of a combination of one ormore of the active compounds with a suppository base. Suitablesuppository bases are, for example, natural or synthetic triglycerides,or paraffin hydrocarbons. In addition, it is also possible to usegelatin rectal capsules which consist of a combination of the activecompounds with a base. Possible base materials include, for example,liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400 (thecompounds are soluble in PEG-400). Aqueous injection suspensions maycontain substances which increase the viscosity of the suspensioninclude, for example, sodium carboxymethyl cellulose, sorbitol, and/ordextran. Optionally, the suspension may also contain stabilizers.

In accordance with one aspect of the present invention, compounds of theinvention are employed in topical and parenteral formulations and areused for the treatment of skin damage, such as that caused by exposureto high levels of radiation, including ultraviolet radiation, heat orchemicals.

One or more additional substances which have therapeutic effects on theskin may also be incorporated in the compositions. Thus, the compositionmay also contain one or more compounds capable of increasing cyclic-AMPlevels in the skin. Suitable compounds include adenosine or a nucleicacid hydrolysate in an amount of about 0.1-1% and papaverine, in anamount of about 0.5-5%, both by weight based on the weight of thecomposition. Also suitable are β-adrenergic agonists such asisoproterenol, in an amount of about 0.1-2% or cyclic-AMP, in an amountof about 0.1-1%, again both by weight based on the weight of thecomposition. Other suitable types of additional active ingredients whichmay be incorporated in the compositions of this invention include anycompounds known to have a beneficial effect on skin. Such compoundsinclude retinoids such as Vitamin A, in an amount of about 0.003-0.3% byweight and chromanols such as Vitamin E or a derivative thereof in anamount of about 0.1-10% by weight, both based on the weight of thecomposition. Additionally, anti-inflammatory agents and keratoplasticagents may be incorporated in the cosmetic composition. A typicalanti-inflammatory agent is a corticosteroid such as hydrocortisone orits acetate in an amount of about 0.25-5% by weight, or a corticosteroidsuch as dexamethasone in an amount of about 0.025-0.5% by weight, bothbased on the weight of the composition. A typical keratoplastic agent iscoal tar in an amount of about 0.1-20% or anthralin in an amount ofabout 0.05-2% by weight, both based on the weight of the composition.

The topical compositions of this invention are formulated preferably asoils, creams, lotions, ointments and the like by choice of appropriatecarriers. Suitable carriers include vegetable or mineral oils, whitepetrolatum (white soft paraffin), branched chain fats or oils, animalfats and high molecular weight alcohol (greater than C₁₂). The preferredcarriers are those in which the active ingredient is soluble.Emulsifiers, stabilizers, humectants and antioxidants may also beincluded as well as agents imparting color or fragrance, if desired.Additionally, transdermal penetration enhancers can be employed in thesetopical formulations. Examples of such enhancers can be found in U.S.Pat. Nos. 3,989,816 and 4,444,762.

Creams are preferably formulated from a mixture of mineral oil,self-emulsifying beeswax and water in which mixture the activeingredient, dissolved in a small amount of an oil such as almond oil, isadmixed. A typical example of such a cream is one which includes about40 parts water, about 20 parts beeswax, about 40 parts mineral oil andabout 1 part almond oil.

Ointments may be formulated by mixing a solution of the activeingredient in a vegetable oil such as almond oil with warm soft paraffinand allowing the mixture to cool. A typical example of such an ointmentis one which includes about 30% almond oil and about 70% white softparaffin by weight.

Lotions may be conveniently prepared by dissolving the activeingredient, in a suitable high molecular weight alcohol such aspropylene glycol or polyethylene glycol.

In addition, these compositions may include other medicinal agents,growth factors, wound sealants, carriers, etc., that are known orapparent to those skilled in the art. The compositions of the inventionare administered to a warm-blooded animal, such as human, alreadysuffering from a skin damage, such as a burn, in an amount sufficient toallow the healing process to proceed more quickly than if the host werenot treated. Amounts effective for this use will depend on the severityof the skin damage and the general state of health of the patient beingtreated. Maintenance dosages over a prolonged period of time may beadjusted as necessary. For veterinary uses, higher levels may beadministered as necessary.

In the case of an animal suffering from decreased hair growth, thecompositions of the invention are administered in an amount sufficientto increase the rate of hair growth. Amounts effective for this use willdepend on the extent of decreased hair growth, and the general state ofhealth of the patient being treated. Maintenance dosages over aprolonged period of time may be adjusted as necessary. For veterinaryuses, higher levels may be administered as necessary.

When the compounds are to be administered to plants, they may be appliedto the leaves and/or stems and/or flowers of the plant, e.g. byspraying. The compounds may be spayed in particulate form or dissolvedor suspended in an appropriate carrier, e.g. in water or an oil-wateremulsion. The compounds may also be combined with the soil of the plant.In this embodiment, the compounds are taken up by the roots of theplant.

In a preferred embodiment, the caspase inhibitor is formulated as partof a mouthwash for the treatment, amelioration or prevention of oralmucositis. Such mouthwashes are aqueous solutions of the caspaseinhibitor which may also contain alcohol, glycerin, synthetic sweetenersand surface-active, flavoring and coloring agents. They may also containanti-infective agents such as hexetidine and cetylpyridinium chloride.The mouthwashes may also contain topical anesthetics (e.g. benzocaine,cocaine, dyclonine hydrochloride, lidocaine, proparacaine hydrochlorideor teracaine hydrochloride), for example, for relieving pain ofradiation or chemotherapy-induced sores. The mouth washes may haveeither acidic or basic pH. See Remington's Pharmaceutical Sciences, A.R. Gennaro (ed.), Mack Publishing Company, pp. 1045, 1046, 1526 and 1965(1990).

In another preferred embodiment, the caspase inhibitor is formulated asan oral formulation which is capable of coating the gastrointestinalsurfaces for the treatment, amelioration or prevention ofgastrointestinal mucositis. Examples of gastrointestinal mucositisinclude esophageal mucositis, gastric mucositis, and intestinalmucositis. Such formulations may comprise gastric antacids such asaluminum carbonate, aluminum hydroxide gel, bismuth subnitrate, bismuthsubsalicylate, calcium carbonate, dihydroxyaluminum sodium carbonate,magaldrate, magnesium carbonate, magnesium hydroxide, magnesium oxide,sodium bicarbonate, milk of bismuth, dihydroxyaluminum aminoacetate,magnesium phosphate, magnesium trisilicate and mixtures thereof. Otheradditives include without limitation H₂-receptor antagonists,digestants, anti-emetics, adsorbants, and miscellaneous agents. SeeRemington's Pharmaceutical Sciences, A. R. Gennaro (ed.), MackPublishing Company, pp. 774-778 (1990).

Chemotherapy agents such as cisplatin and radiation therapy often induceearly and late onset emesis in the patient. Thus, in one embodiment anantiemetic is coadminstered together with the caspase inhibitor to avoidemesis and retain contact of the caspase inhibitor with thegastrointestinal tract. Examples of such antiemetics include withoutlimitation compounds that block the dopaminergic emetic receptors suchas metoclopramide and trimethobenzamide, and cannabinoids.Metoclopramide may be administered orally prior to and/or duringchemotherapy/radiation therapy/caspase inhibitor therapy to prevent theearly emesis response and then later by intranasal administrationaccording to U.S. Pat. Nos. 5,760,086 and 4,536,386 to prevent delayedonset emesis. During the period after chemotherapy/radiation therapy,both the caspase inhibitor and the antiemetic may be coadministered totreat, ameliorate or prevent gastrointestinal mucositis.

In a further embodiment, the caspase inhibitor may be formulated as anIV injectable solution for the treatment, amelioration or prevention ofbone marrow cell death.

The compositions may be administered to a warm-blooded animal, such ashuman, already suffering from chemotherapy or radiation therapy-inducednon-cancer cell death, or, more preferably, before or during therapywith chemotherapy or radiation.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered in clinical therapy and which are obvious to those skilledin the art are within the spirit and scope of the invention.

EXAMPLE 1 2-(Z-Amino)benzoyl-Asp-fmk

Step A. 2-Z-Aminobenzoic acid. To a solution of 2-aminobenzoic acid(0.30 g, 2.2 mmol) in pyridine (2 mL) was added benzyl chloroformate(0.6 mL, 4.2 mmol) at 0° C. The mixture was then stirred at roomtemperature for 1 h, diluted with EtOAc (50 mL), washed with 2N HCl,water and brine, dried over Na₂SO₄ and concentrated in vacuo. The crudesolid was washed with 4:1 hexane/EtOAc twice and dried in vacuo to yieldthe title compound as a white solid (270 mg, 1.0 mmol, 45%). ¹H NMR(DMSO-d₃): δ 11.49 (br s, 1H), 8.22 (d, J=7.5, 1H), 7.95 (d, J=7.5, 1H),7.51 (t, J=7.5, 1H), 7.39-7.32 (m, 5H), 7.05 (d, J=7.5, 1H), 5.15 (s,2H).

Step B. tert-Butyl5-fluoro-3-[2-Z-aminobenzoylamido]-4-hydroxypentanoate. A mixture of2-Z-aminobenzoic acid (90 mg, 0.33 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 61mg, 0.40 mmol), 1-hydroxybenzotriazole hydrate (HOBT, 73 mg, 0.38 mmol),dimethylaminopyridine (DMAP, 22 mg, 0.18 mmol) and tert-butyl3-amino-5-fluoro-4-hydroxypentanoate (79 mg, 0.38 mmol) in THF (10 mL)was stirred at room temperature for 20 h, diluted with 1:1 hexane/EtOAc(75 mL), washed with water, 2N HCl, water, 2N NaOH and brine, dried overNa₂SO₄ and cocentrated in vacuo. The residue was purified bychromatography (3:2 Hexane/EtOAc) to yield the title compound as ayellow hydroscopic solid (52 mg, 0.11 mmol, 33%).

Step C. 2-(Z-Amino)benzoyl-Asp(OBu-t)-fmk. A mixture of periodinane(0.41 g, 0.97 mmol) and tert-butyl5-fluoro-3-[2-Z-aminobenzoylamido]-4-hydroxypentanoate (52 mg, 0.11mmol) in dichloromethane (15 mL) was refluxed for 20 h, cooled to roomtemperature, and 25 mL of saturated sodium bicarbonate aqueous solutioncontaining 1.0 g of Na₂S₂O₃ was added. The resulting mixture was stirredfor 2 h, diluted with 1:1 hexane/EtOAc (75 mL), washed with water andbrine, dried over Na₂SO₄ and concentrated in vacuo. The residue waspurified by chromatography (3:2 hexane/EtOAc) to yield the titlecompound as a hydroscopic yellow solid (45 mg, 0.10 mmol, 91%). ¹H NMR(CDCl₃): δ 10.48 (s, 1H), 8.42 (d, J=8.7, 1H), 7.51-7.33 (m, 7H), 7.07(m, 1H), 5.30-4.98 (m, 1H), 5.21 (s, 2H), 3.11-2.83 (m, 2H), 1.44 (s,9H).

Step D. 2-(Z-Amino)benzoyl-Asp-fmk To a solution of2-(Z-Amino)benzoyl-Asp(OBu-t)-fmk (45 mg, 0.10 mmol) in 5 mL of CH₂Cl₂was added 1 mL of TFA. The resulting solution was allowed to stir at rtfor 1 hr, diluted with EtOAc (75 ml), washed with water, aqueous Na₂HPO₄to pH 5 and then brine, dried over Na₂SO₄ and concentrated in vacuo togive the title compound as a white solid, (15 mg, 0.037 mmol, 38%). ¹HNMR (DMSO-d₆): δ 10.63 (s, 1H), 9.08 (s, 1H), 8.14 (d, J=7.8, 1H), 7.76(d, J=7.8, 1H), 7.54 (t, J=7.8, 1H), 7.41-7.35 (m, 5H), 7.15 (t, J=7.8,1H), 5.16 (s, 2H), 5.26-4.95 (m, 2H), 4.83 (m, 1H), 3.00-2.64 (m, 2H).

EXAMPLE 2 2-(Z-Amino)-6-methylbenzoyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from 2-amino-6-methylbenzoic acid. ¹H NMR (DMSO-d₆): δ 8.85 (s, 1H),8.68 (s, 1H), 7.49-7.01 (m, 8H), 5.12 (s, 2H), 4.82 (m, 1H), 5.26-4.95(m, 2H), 3.00-2.64 (m. 2H), 2.26 (s, 3H).

EXAMPLE 3 2-(Z-Amino)-5-methylbenzoyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from 2-amino-5-methylbenzoic acid. ¹H NMR (DMSO-d₆): δ 10.48 (s, 1H),9.10 (d, J=9, 1H), 8.00 (d, J=8.7, 1H), 7.89 (s, 1H), 7.58 (s, 1H),7.41-7.34 (m, 5H), 5.14 (s, 2H), 4.83 (m, 1H), 5.39-4.41 (m, 2H),2.94-2.80 (m. 2H), 2.30 (s, 3H).

EXAMPLE 4 2-(Z-Amino)-3-methylbenzoyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from 2-amino-3-methylbenzoic acid. ¹H NMR (DMSO-d₆): δ 9.05 (s, 1H),8.73 (s, 1H), 7.38-7.20 (m, 8H), 5.08 (s, 2H), 4.72 (m, 1H), 5.32 (bs,2H), 2.81-2.66 (m. 2H), 2.21 (s, 3H).

EXAMPLE 5 2-(Z-Amino)-3-methoxybenzoyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from 2-amino-3-methoxybenzoic acid. ¹H NMR (DMSO-d₆): δ 8.73 (bs, 1H),8.64 (bs, 1H), 7.57-7.06 (m, 9H), 5.06 (s, 2H), 4.72 (bs, 1H), 5.26-4.97(m, 2H), 3.78 (s, 3H), 2.78-2.66 (m. 2H).

EXAMPLE 6 2-(Z-Amino)-5-fluorobenzoyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from 2-amino-5-fluorobenzoic acid. ¹H NMR (DMSO-d₆): δ 10.40 (bs, 1H),9.13 (bs, 1H), 8.07 (q, J=5.1, 1H), 7.61 (d, J=6.6, 1H), 7.46-7.33 (m,6H), 5.15 (s, 2H), 4.81 (bs, 1H), 2.84-2.72 (m. 2H).

EXAMPLE 7 cis-2-(Z-Amino)cyclohexanecarboxyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from cis-2-aminocyclohexanecarboxylic acid. ¹H NMR (DMSO-d₆): δ 8.28(bs, 1H), 7.39-7.09 (m, 5H), 4.98 (s, 2H), 4.52-4.45 (m, 1H), 3.99 (bs,1H), 2.62-2.53 (m, 4H), 1.77-1.23 (m, 8H).

EXAMPLE 8 2-(Z-Amino)-3,5-dimethylbenzoyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from 2-amino-3,5-dimethylbenzoic acid. ¹H NMR (DMSO-d₆): δ 8.05 (s, 1H),7.42-7.17 (m, 8H), 5.15 (s, 2H), 5.19-5.03 (m, 2H), 4.87 (m, 1H), 2.30(s, 3H), 2.26 (s, 3H).

EXAMPLE 9 2-(Z-Amino)-5chlorobenzoyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from 2-amino-5-chlorobenzoic acid. ¹H NMR (DMSO-d₆): δ 10.56 (s, 1H),9.19 (s, 1H), 8.15 (d, J=9.0, 1H), 7.84 (s, 1H), 7.61 (d, J=9.0, 1H),7.41-737 (m, 5H), 5.16 (s, 2H), 4.81 (m, 1H), 5.41-4.80 (m, 2H),2.84-2.73 (m, 2H).

EXAMPLE 10 2-(Z-Amino)-6-chlorobenzoyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from 2-amino-6-chlorobenzoic acid. ¹H NMR (DMSO-d₆): δ 9.17 (d, J=4.2,1H), 8.95 (s, 1H), 7.74-7.24 (m, 8H), 5.50-5.21 (m, 2H), 5.15 (s, 2H),4.85-4.78 (m, 1H), 2.98-2.65 (m, 2H).

EXAMPLE 11 2-(Z-Amino)-4-chlorobenzoyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from 2-amino-3,5-dimethylbenzoic acid. ¹H NMR (DMSO-d₆): δ 10.84 (s,1H), 9.19 (s, 1H), 8.24 (s, 1H), 7.81 (d, J=8.4, 1H), 7.42-7.24 (m, 6H),5.18 (s, 2H), 5.25-5.20 (m, 2H), 4.82 (m, 1H), 2.94-2.63 (m, 2H).

EXAMPLE 12 3-(Z-Amino)thiophene-2-carboxyl-Asp-fmk

Step A. 3-(Z-Amino)thiophene-2-carboxylic acid. The mixture of methyl3-aminothiophene-2-carboxylate (0.2 g, 1.27 mmol) in 2N NaOH (10 mL) washeated at 90° C. for 15 min, then cooled to 0° C. To the resultingsolution was added benzyl chloroformate (1.5 mL, 10.5 mmol) and THF (10mL). The mixture was then stirred at room temperature for 1 h, washedwith 3:1 hexane: ethyl acetate (2×15 mL). The aqueous phase wasacidified with 2N HCl to pH˜1-2, extracted with ethyl acetate (3×15 mL),washed with water and brine, dried over Na₂SO₄ and concentrated in vacuoto yield the title compound as a white solid (70 mg).

The title compound was then prepared in three steps as described inExample 1 (B-D). ¹H NMR (DMSO-d₆): δ 10.43 (s, 1H), 8.74 (s, 1H), 7.81(d, J=5.4, 1H), 7.73 (d, J=5.4, 1H), 7.44-7.35 (m, 5H), 5.18 (s, 2H),5.32-5.04 (m, 2H), 4.79 (m, 1H), 2.88-2.67 (m, 2H).

EXAMPLE 13 3-(Methoxycarbonylamino)thiophene-2-carboxyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 12from methyl 3-aminothiophene-2-carboxylate and methyl chloroformate. ¹HNMR (DMSO-d₆): δ 10.34 (s, 1H), 8.70 (s, 1H), 7.79 (d, J=5.7, 1H), 7.72(d, J=5.7, 1H), 5.31-4.80 (m, 3H), 3.70 (s, 3H), 2.96-2.73 (m, 2H).

EXAMPLE 14 Cis-2-(Z-Amino)cyclopentanecarboxyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from cis-2-aminocyclopentanecarboxylic acid. ¹H NMR (DMSO-d₆): δ 8.35(s, 1H), 7.34-7.28 (m, 5H), 7.09 (m, 1H), 5.13-4.50 (m, 5H), 4.11 (m,1H), 2.81 (m, 1H), 2.73-2.51 (m, 2H), 1.91-1.40 (m, 6H).

EXAMPLE 15 Trans-2-(Z-Amino)cyclohexanecarboxyl-Asp-fmk

The title compound was prepared in four-steps as described in Example 1from trans-2-aminocyclohexanecarboxylic acid. ¹H NMR (DMSO-d₆): δ 12.48(s, 1H), 8.26-8.15 (m, 1H), 7.38-7.17 (m, 5H), 5.18-4.47 (m, 5H),2.67-2.50 (m, 2H), 2.19 (m, 1H), 1.83-1.06 (m, 10H).

EXAMPLE 16 Z-Glu-(2-aminobenzoyl)-Asp-fmk

Step A. Z-Glu(OBu-t)-2-aminobenzoic acid. To a solution ofZ-Glu(OBu-t)OH (272 mg, 0.81 mmol) in THF (5 mL) was addedN-methylmorpholine (110 μL, 1.1 mmol) at −45 ° C., followed by isobutylchloroformate (105 μL, 0.81 mmol). The mixture was stirred at −45 ° C.for 30 min. and a solution of anthranllic acid (127 mg, 0.93 mmol) inTHF (5 mL) was added, followed by more N-methylmorpholine (200 μL, 1.82mmol). The resulting mixture was stirred overnight and the cooling bathwas allowed to slowly warm to room temperature. After diluted with EtOAc(100 mL), the mixture was washed with 2N HCl, water, saturated NaHCO₃,water, 2N HCl, water and brine, dried over Na₂SO₄ and concentrated invacuo to yield the product as a highly hydroscopic white solid (330 mg,0.72 mmol, 89%). ¹H NMR (DMSO-d₆): δ 1.79 (s, 1H), 8.59 (d, J=8.6, 1H),8.00 (t, J=6.0, 1H), 7.60 (t, J=8.6, 1H), 7.39-7.31 (m, 5H), 7.17 (t,J=7.8, 1H), 5.16-4.99 (m, 2H), 4.08 (m, 1H), 2.33 (m, 2H), 2.08-1.75 (m,2H), 1.38 (s, 9H).

Step B. tert-Butyl5-fluoro-3-[Z-Glu(OBu-t)-(2-aminobenzoyl)amido]-4-hydroxypentanoate. Amixture of Z-Glu(OBu-t)-2-aminobenzoic acid (330 mg, 0.72 mmol), EDCI(129 mg, 0.67 mmol), HOBT (104 mg, 0.68 mmol), DMAP (46 mg, 0.38 mmol)and tert-butyl 3-amino-5-fluoro-4-hydroxypentanoate (136 mg, 0.66 mmol)in THF (6 mL) was stirred at room temperature for 20 h. After dilutedwith 1:1 hexane/EtOAc (75 mL), the mixture was washed with water, 2NHCl, water, 2N NaOH and brine, dried over Na₂SO₄ and concentrated invacuo. The residue was purified by chromatography (3:1 then 3:2hexane/EtOAc) to yield the title compound as a white solid (45 mg, 0.068mmol, 10%).

Step C. Z-Glu(OBu-t)-[2-aminobenzoyl]-Asp(OBu-t)-fmk The title compoundwas synthesized by a similar procedure as described in Step C, Example 1in 58% yield.

Step D. Z-Glu-[2-aminobenzoyl]-Asp-fmk. The title compound wassynthesized by a similar procedure as described in Step D, Example 1 in14% yield. ¹H NMR (DMSO-d₆): δ 1.46 (s, 1H), 9.18 (s, 1H), 8.57-7.20 (m,6H), 5.36-4.84 (m, 5H), 4.04 (br s, 1H), 2.95-1.81 (m, 6H).

EXAMPLE 17 Z-Val-(2-Aminobenzoyl)-Asp-fmk

The title compound was synthesized as described in Example 16 fromZ-Val. ¹H NMR (DMSO-d₆): δ 1.34-11.25 (m, 1H), 9.17-7.17 (m, 11H),5.42-4.30 (m, 5H), 3.95-3.75 (m, 1H), 2.95-2.57 (m, 2H), 1.92 (m, 1H),0.91-0.84 (m, 6H).

EXAMPLE 18 2-(Z-Amino)benzoyl-Asp-DCB-methylketone

Step A. Z-Asp(OBu-t)-DCB-methylketone To a solution ofZ-Asp(OBu-t)-bromomethylketone (500 mg, 1.24 mmol) in DMF (10 ml) wasadded potassium fluoride (320 mg, 5.50 mmol), and 2,6-dichlorobenzoicacid (348 mg, 1.82 mmol). The mixture was stirred at room temperaturefor 12 h, and then was diluted with 25 ml of ethyl acetate, washed withaqueous NH₄Cl and brine, dried over Na₂SO₄ and concentrated in vacuo.The title compound was obtained as white solid (0.78 g, 2.62 mmol, 69%).¹H NMR (CDCl₃): 7.34 (m, 8H), 5.96 (d, J=8.7, 1H), 5.21 (d, J=6.6, 2H),5.16 (s, 2H), 4.70 (m, 1H), 2.88 (m, 2H), 1.27 (s, 9H).

Step B. Asp(OBu-t)-DCB-methylketone-HCl To a solution ofZ-Asp(OBu-t)-DCB-methylketone (572 mg, 1.14 mmol) in ethanol (15 ml) wasadded Pd/C (50 mg) and 6N HCl (0.2 ml). The mixture was stirred at roomtemperature under H₂ atmosphere (1 atm) for 12 h, then it was filteredand concentrated. The title compound was obtained as pale white solid(416 mg, 1.04 mmol, 90%). ¹H NMR (CDCl₃): 7.27 (m, 3H), 5.28 (m, 2H),4.94 (m, 1H), 3.27 (m, 2H), 1.42 (s, 9H).

Step C. 2-(Z-Amino)benzoyl-Asp(OBu-t)-DCB-methylketone To a solution of2-(Z-amino)benzoic acid (140 mg, 0.52 mmol) in THF (5 mL) was addedN-methylmorpholine (65 μL, 0.59 mmol), followed by 2-methylpropylchloroformate (70 μL, 0.54 mmol) at −45° C. After 30 min., a solution ofAsp(OBu-t)-DCB-methylketone-HCl (121 mg, 0.27 mmol) in THF (5 mL) wasadded to the solution. The resulting mixture was further stirredovernight and the cooling bath was allowed to slowly warm to roomtemperature. It was then diluted with 1:1 hexane/EtOAc (100 mL), washedwith water, 2N NaOH and brine, dried over Na₂SO₄ and concentrated invacuo. The residue was purified by chromatography (3:1 hexane/EtOAc) toyield the title compound as a white solid (365 mg, 0.05 mmol, 19%). ¹HNMR (CDCl₃): 10.90 (s, 1H), 8.49 (d, J=7.5, 1H), 7.87 (dd, J=8.1, 1.8,1H), 7.56-7.32 (m, 9H), 7.08 (t, J=6.9, 1H), 5.23 (s, 2H), 5.28 (m, 1H),4.90 (d, J=1.8, 2H), 3.16-2.91 (m, 1H), 1.43 (m, 9H).

Step D. 2-(Z-Amino)benzoyl-Asp-DCB-methylketone A solution of2-(Z-amino)benzoyl-Asp(OBu-t)-DCB-methylketone (35 mg) and TFA (1 mL) inmethylenechloride (3 mL) was stirred at room temperature for 2 h. Themixture was diluted with EtOAc (70 mL), washed with saturated Na₂HPO₄ topH˜5, and further washed with water, and brine, dried over Na₂SO₄ andconcentrated in vacuo to yield the title compound as a white solid (10mg, 0.016 mmol, 33%). ¹H NMR (CDCl₃): 10.94 (s, 1H), 8.49 (d, J=8.4,1H), 7.87 (dd, J=8.1, 1.5, 1H), 7.53 (m, 1H), 7.45-7.33 (m, 8H), 7.07(m, 1H), 5.22 (s, 2H), 5.10-5.07 (m, 1H), 4.90 (m, 2H), 3.11 (dd, J=8.4,19.0, 1H), 2.95 (dd, J=1.8, 19.0, 1H)).

EXAMPLE 19 Methoxycarbonyl- Val-(2-aminobenzoyl)-Asp-fmk

Step A. tert-Butyl5-fluoro-3-(2-aminobenzoylamido)-4-hydroxypentanoate.HCl. A mixture oftert-butyl 5-fluoro-3-(2-Z-aminobenzoylamido)-4-hydroxypentanoate (80mg, 0.174 mmol), Pd-C (23mg) and 6N HCl (0.087 mL) in ethanol (5 mL) wasstirred under hydrogen atmosphere at room temperature for 2 h. Themixture was filtered and the solvent was evaporated to yield the titleproduct. It was used in next step without further purification.

Step B. tert-Butyl5-fluoro-3-(methoxycarbonyl-Val-2-aminobenzoylamido)-4-hydroxypentanoate.To a solution of methoxycarbonyl-Val-OH (31 mg, 0.1 7 mmol) in THF (5mL) was added N-methylmorpholine (38 μL, 0.34 mmol) at −45° C., followedby isobutyl chloroformate (45 μL, 0.034 mmol). The mixture was stirredat −45° C. for 30 min and a solution of tert-butyl5-fluoro-3-(2-aminobenzoylamido)-4-hydroxypentanoate.HCl in THF (5 mL)was added, followed by more N-methylmorpholine (50 μL, 0.45 mmol). Theresulting mixture was stirred overnight and the cooling bath was allowedto slowly warm to room temperature. After dilution with ethyl acetate(50 mL), the mixture was washed with water and brine, dried over Na₂SO₄and concentrated in vacuo. The residue was purified by chromatography(3:2 hexane: ethyl acetate) to yield the title compound as a whitehydroscopic solid (20 mg, 0.041 mmol, 24%). ¹H NMR (CDCl₃): δ 1.36-11.24(m, 1H), 8.54 (d, J=8.7, 1H), 7.55-7.05 (m, 4H), 5.40 (m, 1H), 4.89-3.85(m, 6H), 3.71 (d, J=1.8, 3H), 2.84-2.61 (m, 2H), 2.29 (m, 1H), 1.46-1.44(m, 9H), 1.05-0.98 (m, 6H).

Step C, D. Methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk The titlecompound was synthesized with a similar procedure as described in Step Cand D of example 1. ¹H NMR (DMSO-d₆): δ 2.52 (s, 1H), 11.27 (d, J=6.0,1H), 9.18 (m, 1H), 7.88-7.54 (m, 4H), 7.20 (t, J=7.5, 1H), 5.39-4.44 (m,3H), 3.83 (m, 1H), 3.57 (d, J=2.4, 3H), 2.96-2.65 (m, 2H), 2.18 (m, 1H),0.92 (t, J=6.3, 6H).

EXAMPLE 20 Enzyme Activity

The activity of 2-(Z-amino)benzoyl-Asp-fmk as an inhibitor of caspase-3was measured in a fluorometric enzyme assay. Enzyme activity wasmeasured using synthetic peptide substrates attached to a fluorogenicleaving group. Cleavage of the synthetic substrate by the enzyme resultsin a fluorescent signal which is read in a spectrofluorometer or in afluorometric microtiter plate reader.

Twelve concentrations of the testing compound ranged from 30 pM to 10 μMwere tested in the enzyme assay. The enzyme reaction was conducted inthe presence of 2 ng rCaspase 3 (purchased from PharMingen, a Bectondivision company, San Diego, Calif.), various concentrations of testingcompound, 10 μM caspase 3 substrate Ac-DEVD-AMC (SEQ ID NO:1) (purchasedfrom Quality Controlled Biochemicals, Inc., Hopkinton, Mass.) andcaspase buffer (20 mM PIPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1%CHAPS and 10% sucrose, pH 7.2) in a total volume of 100 μL. The enzymereaction was carried out in a 96-well plate and incubated at 37° C. for30 minutes. The plate was then read with a fluorescence plate reader(EG&G WALLAC 1420-002) using excitation filter at 355 nm/emission filterat 460 nm. The data was analyzed using GraphPrism software to give anIC₅₀ value of 0.2 μM.

Having now fully described this invention, it will be understood bythose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents, patent applications and publicationscited herein are fully incorporated by reference herein in theirentirety.

1 1 4 PRT Homo sapiens MOD_RES (1)(1) N-terminal acetyl 1 Asp Glu ValAsp 1

What is claimed is:
 1. A compound having the Formulae I or II or III:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: R₁ isan optionally substituted alkyl or hydrogen; R₃ is an N-protectinggroup; R₂ is hydrogen or optionally substituted alkyl; Q is anoptionally substituted saturated or partially saturated carbocycle orheterocycle; X is a peptide of 1-4 amino acids or a bond; Y is a peptideof 1-4 amino acids or a bond; A is CR₆ or nitrogen; B is CR₇ ornitrogen; C is CR₈ or nitrogen; D is CR₉ or nitrogen; provided that notmore than two of A, B, C or D is nitrogen; and R₆-R₉ independently arehydrogen, halo, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀aryl(C₂-C₆)alkenyl, C₆-C₁₀ aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl,nitro, amino, cyano, C₁-C₆ acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆alkoxy, alkylthio, or carboxy; or one of R₆ and R₇, or R₇ and R₈, or R₈and R₉ are taken together with the carbon atoms to which they areattached to form a carbocycle or heterocycle; E is C₁₄, nitrogen, oxygenor sulfur; F is C₁₅, nitrogen, oxygen or sulfur; G is C₁₆, nitrogen,oxygen or sulfur; provided that only one of E, F, G is nitrogen, oxygenor sulfur and R₁₄-R₁₆ are independently hydrogen, halo, C₁-C₆ haloalkyl,C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy;or one of R₁₄ and R₁₅, or R₁₅ and R₁₆, are taken together with thecarbon atoms to which they are attached to form a carbocycle orheterocycle.
 2. A compound according to claim 1, wherein R₃ ist-butyloxycarbonyl, acetyl or benzyloxycarbonyl.
 3. A compound accordingto claim 1, wherein R₁ is H, Me, Et or acetoxymethyl.
 4. A compoundaccording to claim 1, wherein R₂ is hydrogen, fluoromethyl,acyloxymethyl, arylacyloxymethyl or aminomethyl.
 5. A compound accordingto claim 1, wherein X is a bond.
 6. A compound according to claim 1,wherein A, B, C and D are CH.
 7. A compound according to claim 1,wherein A is nitrogen, and B, C and D are CH.
 8. A compound according toclaim 1, wherein G is sulfur, and E and F are CH.
 9. A compoundaccording to claim 1, wherein Q is cyclohexyl or cyclopentyl.
 10. Acompound according to claim 1, wherein said compound is selected fromthe group consisting of: 2-(Z-amino)benzoyl-Asp-fmk,2-(Z-amino)-3-methylbenzoyl-Asp-fmk,2-(Z-amino)-3,S-dimethylbenzoyl-Asp-fmk,2-(Z-amino)-4-chlorobenzoyl-Asp-fmk,2-(Z-amino)-5-chlorobenzoyl-Asp-fmk,2-(Z-amino)-5-fluorobenzoyl-Asp-fmk,2-(Z-amino)-6-fluorobenzoyl-Asp-fmk,cis-2-(Z-amino)-cyclohexanecarboxy-Asp-fmk,2-(Z-amino)-5-methylbenzoyl-Asp-fmk,2-(Z-amino)-6-methylbenzoyl-Asp-fmk,2-(Z-amino)-6-chlorobenzoyl-Asp-fmk,2-(Z-amino)-3-methoxybenzoyl-Asp-fmk,3-(Z-amino)thiophene-2-carboxyl-Asp-fmk,3-(methoxycarbonylamino)thiophene-2-carboxyl-Asp-fmk,cis-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,trans-2-(Z-amino)cyclopentanecarboxyl-Asp-fmk,2-(Z-amino)benzoyl-Asp-DCB-methylketone,methoxycarbonyl-Val-(2-aminobenzoyl)-Asp-fmk,Z-Glu-(2-aminobenzoyl)-Asp-fmk, and Z-Val-(2-aminobenzoyl)-Asp-fmk. 11.A pharmaceutical composition, comprising a compound of claim 1, and apharmaceutically acceptable carrier.
 12. A compound according to claim1, wherein said compound has the Formula IV:

or a pharmaceutically acceptable salt or prodrug thereof, wherein R₂ ishydrogen or optionally substituted alkyl, wherein the substituent ishalo, hydroxy, alkoxy, aryloxy, alkylthio, arylthio, amino, acyloxy, orarylacyloxy; R₆-R₉ independently are hydrogen, halo, C₁-C₆ haloalkyl,C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, C₆-C₁₀ aryl(C₂-C₆)alkenyl, C₆-C₁₀aryl(C₂-C₆)alkynyl, C₁-C₆ hydroxyalkyl, nitro, amino, cyano, C₁-C₆acylamino, hydroxy, C₁-C₆ acyloxy, C₁-C₆ alkoxy, alkylthio, or carboxy;or one of R₆ and R₇, or R₇ and R₈, or R₈ and R₉ are taken together withthe carbon atoms to which they are attached to form a carbocycle orheterocycle, selected from the group consisting of —OCH₂O—, —OCF₂O—,—CH₂)₃—, —CH₂)₄—, —OCH₂CH₂O—, —CH₂N(R₁₃)CH₂—, —CH₂CH₂N(R₁₃)CH₂—,—CH₂N(R₁₃)CH₂CH₂— and —CH═CH—CH═CH—; wherein R₁₃ is hydrogen, alkyl orcycloalkyl; R₁₀ is hydrogen, C₁-C₆ alkoxy, C₁-C₆ alkyl, C₁-C₆ haloalkyl,C₆-C₁₀ aryl, C₄-C₇ cycloalkyl, C₆-C₁₀ aryl(C₁-C₆)alkyl, benzyloxy,substituted benzyloxy, or NR₁₁R₁₂; wherein R₁₁ and R₁₂ independently arehydrogen, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, C₄-C₇ cycloalkyl,C₆-C₁₀ aryl(C₁-C₆)alkyl, or R₁₁, and R₁₂ are combined to form aheterocyclic ring system selected from the group consisting ofpyrrolidine, piperidine, piperazine, and morpholine.
 13. A compoundaccording to claim 12, wherein R₂ is hydrogen, fluoromethyl,acyloxymethyl, arylacyloxymethyl or aminomethyl.
 14. A compoundaccording to claim 12, wherein R₁₀ is benzyloxy.
 15. A compoundaccording to claim 12, wherein R₁ is H, Me or acetoxymethyl.
 16. Acompound according to claim 12, wherein X is a peptide of 1-2 aminoacids or a bond.